1. Field of the Invention
This invention relates to the identification, isolation and use of markers and antibodies which recognize these markers in the diagnosis of invasive prostatic neoplasia in humans, and to diagnostic aids for screening biological samples for evidence of invasive prostatic neoplasia.
2. Description of the Background
The prostate, an organ of the mammalian male urogenital system, is located at the base of the bladder surrounding the urethra. Although encapsulated, the walnut-sized prostate can be divided into five lobes, the posterior, middle, and exterior lobes and two lateral lobes. Histological examination reveals that the prostate is a highly microvascularized gland comprising fairly large glandular spaces lined with epithelium. The majority of fluid of the male ejaculate is supplied by this gland and the seminal vesicles.
The prostate is an endocrine-dependent organ which responds to both the major male hormone, testosterone, and the major female hormones, estrogen and progesterone. In particular, the testicular androgen is believed important for prostate growth and development because, in both humans and other animals, castration leads to prostate atrophy and an absence of any incidence of prostatic carcinoma.
There are two major neoplasia of the prostate, benign enlargement of the prostate or nodular hyperplasia (also called benign prostatic hyperplasia (BPH) or benign prostatic hypertrophy), and prostatic carcinoma. Nodular hyperplasia is very common in men over the age of 50. It is characterized by the presence of a number of large distinct nodules in the periurethral area of the prostate. Although benign, these nodules can produce obstruction of the urethra causing nocturia, micturition, and difficulty in starting and stopping a urine stream upon voiding the bladder. Occasionally, catheterization is required and even surgery. In the more extreme cases, secondary changes in the bladder can occur such as hypertrophy, acute retention with secondary urinary tract involvement, azotemia and uremia. Although all of these changes of the prostate may suggest pre-malignancy, there is as yet no direct association between nodular hyperplasia and prostatic carcinoma.
Carcinoma of the prostate is the most common form of cancer in human males with upwards of one third of those cases being fatal. In the more aggressive forms, transformed prostatic tissue escapes from the prostate capsule invading locally and throughout the bloodstream. Local invasions typically involve the seminal vesicles, the base of the urinary bladder, and the urethra. Hematogenous spread occurs primarily to the bones and lymph nodes, but can include massive visceral invasion as well. Histologically, most lesions are adenocarcinomas with well-defined gland patterns, but the more typical malignancy patterns associated with the very aggressive cancers are also common. Except in rare instances, all forms of prostatic carcinoma originate in the peripheral zone of the gland which is palpable upon rectal examination.
Prostatic carcinomas are graded and staged by number and letter according to histological criteria, the arrangement and appearance of malignant glands, and the degree of anaplasia of the cancerous cells. Stage A1 tumors include the incidental or clinically unsuspected cancers. These are detected in autopsy and rarely pose a problem to the patient. Stage B tumors are detectable by rectal digital examination and are also confined to the prostate. Tumors classified as B1, B2, and so on, indicate increasing severity of tumor formation. These tumors are fairly common in older men who begin to show signs and symptoms characteristic of some form of prostatic carcinoma. Stage C tumors have breached the prostate capsule and may or may not have invaded the surrounding tissues such as the seminal vesicles. Those tumors which have seminal vesicle involvement show an 80% correlation with lymph node involvement (C2). Stage D tumors have distinct metastases and a 100% correlation with lymph node involvement. Over 75% of patients with prostatic carcinoma show signs of stage C or D type development with significant urinary tract involvement. Only 5-10% of stage A patients, of those who have been followed for 8-10 years, develop stage C or D type prostatic carcinoma although the probability increases for patients who first :present at a fairly young age. Young males with nodular hyperplasia are typically recommended for surgery or more aggressive endocrine therapy.
Little is known about the causes of prostatic carcinoma, but there are at least three confirmed risk factors--age, race and endocrine system. As discussed, the incidence of all forms of prostatic neoplasia is very high in men over 50. In the 45-49 year old age group the incidence is about 4.8 per one hundred thousand men and increases to 513 between the ages of 70 to 75. The incidence of latent carcinoma is higher still. Over 30% of prostate tissue in autopsied males over 50 shows some sign of latent carcinoma.
The second risk factor, race, is fairly strong. Among white males in the United States the incidence of prostatic neoplasia in those over 50 is about 58 per one hundred thousand men. The rate increases to about 95 per one hundred thousand in black males whereas in oriental males, prostatic neoplasia is rather rare at about 3 to 4 per one hundred thousand in one study performed in Hong Kong. The exact reason for this distribution is unclear. Although environmental effects should not be discounted, epidemiology points to a strong genetic influence.
The final risk factor, the endocrine system, may be the most important. Although, no direct link has been established between absolute or relative levels of any hormone and neoplasia of the prostate, the evidence for some form of hormonal regulation is convincing. First, in both humans and dogs, the only other mammal known to develop hyperplasia with aging, nodular hyperplasia or full-blown carcinoma of the prostate only develop in the presence of intact testes. Secondly, in castrated young dogs it is possible to induce nodular hyperplasia by administering of androgen and estradiol, suggesting that hormones produced by the testes are required for prostate development. Further, there is some evidence that dihydrotestosterone, which is derived from testosterone, may be the ultimate mediator of cell growth. Prostate cells of the epithelium are covered with dihydrotestosterone receptors which increase in number in the presence of estrogen. In men and dogs, plasma testosterone levels decrease and estradiol levels increase with age. This alteration shifts the hormonal balance of the cells and possibly sensitizes the prostate for transformation. At the very least, it appears that androgens are required to maintain the viability of prostate epithelium from which most carcinomas derive.
Yearly rectal examination is very useful for the early detection of prostatic neoplasia. This detection method is fairly simple and straightforward. However, it is subject to bias and not very well standardized. At the earliest, it can only detect stage B carcinoma and has no capacity to determine whether stages C or D are developing. Further, the digital rectal exam is not very sensitive. Approximately 30-60% of men have a prostatic neoplasia that cannot be detected by the physician, which is further complicated by the fact that these men usually present with no symptoms at all. A number of new techniques look promising. These include ultrasound and other methods of noninvasive detection such as positron emission tomography (PET). These methods are limited to the detection of formed tumors and are unable to detect prostatic carcinoma which is just beginning to invade surrounding tissue.
Chemotherapy, surgery or radiotherapy is the treatment of choice for stage A or B prostatic neoplasia. Surgery involves complete removal of the entire prostate, radical prostatectomy, and often removal of the surrounding lymph nodes, lymphadenectomy. Radiotherapy may be either external or interstitial using .sup.125 I and is typically performed in conjunction with surgery. Endocrine therapy is the treatment of choice for more advanced forms. The aim of this therapy is to deprive the prostate cells, and presumably the transformed prostate cells as well, of testosterone. This is accomplished by administering estrogens or synthetic hormones which are agonists of luteinizing hormone-releasing hormone (LHRH). These cellular messengers directly inhibit testicular and organ synthesis and suppress luteinizing hormone (LH) secretion which in turn leads to reduced testosterone secretion by the testes. Despite the advances made in achieving a pharmacologic orchiectomy, the survival rates for those with stage C and D carcinomas are rather bleak. In the short term, the most promising results will be achieved by earlier detection using more sensitive assays.
Carcinoma cell invasion of the seminal vesicles is a very poor prognosis for the patient. As discussed, seminal vesicle involvement frequently correlates with metastases to the lymph nodes and subsequent dissemination throughout the body. Invasion of the seminal vesicles begins with cell multiplication at the base of the prostate. Transformed cells expand within and through the ejaculating duct, localizing in the seminal vesicles near their point of junction with the vas deferens (A. A. Villers et al., J. Urol. 143:1183, 1990). Surprisingly, others have found a relatively low frequency of positive nodes in patients with seminal vesicle invasion, but a comparable prognosis among patients with and without lymph node metastases (E. Mukamel et al., Cancer 59: 1535, 1987). No alternative explanation was proposed. Uncertainty in these results may stem from the fact that the seminal vesicles are not very well defined morphologically or biochemically.
There are two seminal vesicles in man, one located on each side of the urethra posterior to the urinary bladder and superior to the prostate. They are believed to contain two types of epithelial cells, principal or superficial cells, and basal cells. Each gland is open to the urethra and comprises a highly convoluted tube coiled upon itself which, if extended, would be approximately 15 cm in length. The convolutions within each gland impart a honeycomb appearance when viewed under cross section. The internal cells of the individual vesicles are highly interconnected with ridges and folds both circular and longitudinal. Individual cells of the walls contain numerous secretory bodies including golgi vacuoles, electron dense granules and droplets. These bodies secrete a mixture of materials into the lumen of each tubule. Approximately 70% of human ejaculate is composed of this material which contains fructose, citrate, inositol, prostaglandin, choline esters, and a number of soluble proteins. A few of these proteins have been identified as specific to seminal vesicle tissue including semenogelin I, a large molecular weight protein which can be broken down into three subunits of 52 kDa, 71 kDa, and 76 kDa (H. Lilja et al., J. Biol. Chem. 264:1894, 1989), semenogelin II (H. Lilja and A. Lundwall, Proc. Natl. Acad. Sci. USA 89:4559, 1992), lactoferrin or scafferin (A. Hekman and P. Rumke, Fertil. Steril. 20: 312,1969), seminal vesicle specific antigen (SVS A), MHS-5 specific antigen (J. C. Herr et al., J. Reprod. Immunol. 16:99, 1989), rat seminal vesicle specific (SVS) proteins I-VIII (J. Seitz and G. Aumuller, Andrologia 22:25, 1990), B-microseminoprotein (B-MSP) (K. Akiyama et al., Biochim. Biophys. Acta 829:288, 1985), and seminal plasma number 7 antigen (K. Koyama et al., J. Reprod. Immunol. 5:134, 1983). These proteins and antigens are only now being analyzed in detail and some have been cloned by recombinant DNA techniques.
Fairly recently, a number of serum antigens have been characterized as markers for prostatic neoplasia. These markers are useful because they are relatively straightforward to assay using noninvasive procedures and may detect prostatic neoplasia at very early stages of development. Both malignant and normal prostate epithelial cells were found to express a prostate-specific acid phosphatase (PAP) which is detectable in serum by biochemical and other immunological techniques. Elevated PAP levels correlate well with neoplasia that has spread beyond the prostate capsule. Consequently, PAP is a useful serum marker for characterizing the later stages of prostatic neoplasia and also for monitoring the progress of the disease in patients.
Another marker which has proved to be of value is the prostate-specific antigen (PSA), a serine protease found in both normal and neoplastic prostate epithelium. Investigations have determined that there is a direct correlation between serum PSA levels with the size and stage of a tumor. The normal concentration of PSA in men is from 0 to 2.8 ng/ml of serum. In one study, researchers determined that average PSA concentrations in the serum of patients grouped according to severity were proportional to the clinical state of the tumor (T. A. Stamey, et al., N. Engl. J. Med. 317:909, 1987). These authors did not indicate whether PSA levels could be used to determine the pathological stage of carcinoma in individual patients. Concentrations of 40 ng/ml were predictive of advanced stages of disease, but the predictive value of serum concentrations of less then 15 ng/ml were less than clear. PSA titers were only marginally useful to distinguish whether the tumor was contained by or had escaped the prostate. Levels greater than 10 ng/ml were typical in patient groups with more advanced and gland-unconfined carcinomas. However, it was not atypical to find high PSA levels in patient groups with gland-confined hyperplasia.
These theories were partly confirmed in a more recent study which looked at serum PSA levels in 209 men with various stages of prostatic neoplasia (T. E. Osterling et al., J. Urol. 139:766, 1988). These authors determined that PSA levels showed a statistically significant correlation with pathological stages when compared within the various groups. However, the levels were far less useful when looking at patients on an individual basis. There was a large degree of variability between patient groups and a significant number of both false and missed positives. In a rigorous analysis using greater numbers of men and taking into account actual or predicted numbers of carcinoma cells, Partin et al. determined that serum PSA levels were influenced by tumor volume and the stage of differentiation (A. W. Partin et al., J. Urol. 143:747, 1990). Mean antigen levels increased with advanced pathological stage, but this seemed to be related more to overall tumor volume than to any particular stage of the disease. In fact, immunohistochemical studies revealed that higher stage tumors actually produced less PSA, possibly due to the diseased state of the cells. The authors concluded that PSA levels are unreliable for preoperative prediction of the pathological stage of individual patients.
A number of other prostate antigens have since been identified. The most well-studied of these has been the prostatic carcinoma associated complex (PAC) also called the glycoprotein complex (G. L. Wright et al., Int. J. Cancer 47:717, 1991). Although specific for prostatic epithelium, this protein complex of 35-310 kDa antigens was not correlative for the staging of prostatic carcinoma.